The central goal of this project is to understand fundamental mechanisms of human pre-mRNA splicing, a required step in the expression of most eukaryotic genes, as well as the regulation of this process. Specific mechanisms through which exons and introns are correctly identified by the spliceosome will continue to be investigated. These studies will focus on intronic sequences proximal to the splice sites, and on the RNA-binding proteins that recognize them. This project will also investigate an aspect of pre-mRNA splicing fidelity, namely the mechanisms underlying suppression of cryptic splice sites that are only used in the context of mutations elsewhere in a gene. In addition, the network of protein-protein interactions of several splicing-regulatory factors will be characterized. Finaly, the mechanisms underlying the interplay between pre- mRNA splicing and nonsense-mediated mRNA decay, an RNA quality-control process, will continue to be studied. This project will rely on integrative approaches, including biochemical, molecular, proteomics, and bioinformatics techniques, as well as both cell-based and in vitro assays. In addition to obtaining new insights into basic mechanisms of gene expression, these studies will improve the understanding of numerous mutations associated with various genetic diseases, as well as facilitate correct genetic diagnosis and therapeutics development for such diseases. PUBLIC HEALTH RELEVANCE: The proposed studies have broad relevance for the majority of human genetic diseases, because a high proportion of disease-causing mutations are of the type that affect mRNA splicing and stability. This project will result in a better understanding of which mutations cause defective gene expression, and how they do so. In addition, the new findings are expected to facilitate the development of targeted therapeutics to correct certain gene-expression defects.